Then, average fluorescence intensities are measured by conventional flow cytometry analysis and finally a simple calculation is applied to estimate the absolute number of the Halo-tagged protein of interest per cell.
First, a cell line expressing the Halo-tagged protein of interest is grown and labeled side-by-side with our standard line. Here we detail a straightforward flow cytometry-based method to measure the absolute abundance of any Halo-tagged protein in live cells that uses a standard mammalian cell line with a known number of Halo-CTCF proteins recently characterized in our lab. Existing methods to determine absolute protein abundances are labor-intensive and/or require sophisticated experimental and computational infrastructure ( e.g., fluorescence correlation spectroscopy (FCS)-calibrated imaging and quantitative mass spectrometry). Accurate abundance measurements of cellular proteins are required to achieve a quantitative and predictive understanding of any biological process inside the cell.
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